METHOD OF DETECTION OF AUXIN-INDOLE-3-ACETIC ACID IN SOILS BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A sensitive, specific, and precise method for detection of indole-3-acetic acid (IAA) in soils is described. This method involves the use of reverse phase, ion-pair, high performance liquid chromatography (HPLC) to detect IAA produced from L-tryptophan (L-TRP) added to soils. A 20-µL aliquot of the soil extract was injected in the eluent stream consisting of a 60:40 CH3OH:H2O solution containing 0.01M l-heptanesulfonic acid (ion-pair reagent) which was acified to pH 2.5 with H3PO4 and separated with an analytical ultrasphere-ODS (C18) column. Ultraviolet-absorbance (254 nm) was used as the mode of detection. Separation of indole-3-acetamide (IAM) indole-3-pyruvic acid (IPyA) and IAA (identified by mass spectrometry) was achieved only in the presence of the ion-pair reagent. Detection of these 3-substituted indoles in soils incubated with L-TRP suggests that IAA is synthesized by two distinct pathways: (i) TRP is converted to IAA by deaminating to IPyA followed by decarboxylation to indole-3-acetaldehyde (IAAld) which is further oxidized to IAA, and (ii) TRP is decarboxylated to IAM which is hydrolyzed to IAA. The detection limit for IAA by the method described is 50 pg/mL of methanol under standardized conditions. The concentration of IAA can be determined in soil extracts by HPLC in © 1983 Soil Science Society of America.